Methods for measuring the inactivation of pathogens

ABSTRACT

Methods for measuring pathogen inactivation, and in particular, methods for measuring pathogen inactivation in blood and blood products after photochemical decontamination. The methods involve measuring the inhibition of template-dependent enzymatic synthesis of nucleic acid following the addition of compounds that add covalently to nucleic acid.

FIELD OF THE INVENTION

The present invention relates to new methods for measuring theinactivation of pathogens, and in particular, new methods for measuringpathogen inactivation in blood and blood products after adecontamination process.

BACKGROUND

The safety of the blood supply continues to be threatened by thetransmission of pathogens by transfusion. While the threat posed by thehuman immunodeficiency virus (HIV) and the Acquired Immune DeficiencySyndrome (AIDS) is now widely publicized, contamination of bloodproducts with a number of other blood-borne infectious viral agents isof even greater concern. See R. Y. Dodd, In: Transfusion Medicine in the1990's (American Assoc. Blood Banks 1990) (S. J. Nance, ed.). Forexample, in the United States, it is estimated that five to ten percentof multiply transfused recipients develop hepatitis accounting for manythousands of cases annually.

The safety of the blood supply cannot be assured by merely testing theblood for pathogens before transfusion. Most testing relies on thedetection of antibodies to the pathogen in the prospective blood donor.It is now well-documented that infectious agents can be transmitted by"seronegative" blood donors, i.e., donors that have no detectableantibodies to the pathogen. For example, thirteen cases oftransfusion-related AIDS have been reported to the Centers for DiseaseControl (CDC) among recipients of blood that was pretested and foundnegative for antibody to the HIV-1 virus.

Most importantly, routine serologic testing will not detect newinfectious agents. Each time a new infectious agent is identified, a newtest must be designed, developed and approved. During this time,transfusion recipients are at risk. The most dramatic example of this isfound in hemophiliac patients receiving repeated exposure to plasma orclotting factor concentrates. While a test for the HIV virus was beingdeveloped, this patient population was exposed to untested bloodproducts. As a result, greater than ninety percent of this populationnow have serologic evidence of past hepatitis or HIV infection, and manyhave developed overt hepatitis or AIDS.

An alternative approach to eliminate transmission of diseases throughblood products is to develop a means to inactivate pathogens intransfusion products. Several methods have been reported to be effectivein inactivating or eliminating viral agents, HIV in particular, in humanplasma and its derivatives. These methods include thermal inactivation,γ irradiation, laser-UV irradiation, UV irradiation in the presence ofβ-propiolactone, use of organic solvent and detergent combinations andlaser-visible light irradiation in the presence of hematoporphyrin.Unfortunately, most of these methods harm the blood cellular componentsas well.

A more encouraging approach to blood decontamination is thephotochemical decontamination (PCD) process using psoralens. Psoralensare tricyclic compounds formed by the linear fusion of a furan ring witha coumarin. Psoralens can intercalate between the base pairs ofdouble-stranded nucleic acids, forming covalent adducts to pyrimidinebases upon absorption of longwave ultraviolet light (UVA). G. D. Ciminoet al., Ann. Rev. Biochem. 54:1151 (1985). Hearst Et al., Quart. Rev.Biophys. 17:1 (1984). If there is a second pyrimidine adjacent to apsoralen-pyrimidine monoadduct and on the opposite strand, absorption ofa second photon can lead to formation of a diadduct which functions asan interstrand crosslink. S. T. Isaacs et al., Biochemistry 16:1058(1977). S. T. Isaacs et al., Trends in Photobiology (Plenum) pp. 279-294(1982). J. Tessman et al., Biochem. 24:1669 (1985). Hearst et al., U.S.Pat. Nos. 4,124,589, 4,169,204, and 4,196,281, hereby incorporated byreference.

The PCD process has been shown to inactivate a wide range of pathogensin some blood products. See H. J. Alter et al., The Lancet. (ii:1446)(1988). L. Lin et al., Blood 74:517 (1989). G. P. Wiesehahn et al., U.S.Pat. Nos. 4,727,027 and 4,748,120, hereby incorporated by reference,describe the use of PCD process utilizing a combination of8-methoxypsoralen (S-MOP) and irradiation. They show that the PCDprocess can effectively inactivate a number of pathogens, includingintracellular HIV. Furthermore, the harm to the blood product that wouldotherwise occur in the PCD process because of energy transfer, wasspecifically suppressed by limiting the concentration of molecularoxygen.

What remains unclear about the PCD process is the efficiency ofinactivation of free viral and provital genetic material, particularlywith RNA viruses. Because of the low sensitivity and time consumingnature of current biological culture methods, there is no adequate assayat present for measuring pathogen inactivation in blood products.

SUMMARY OF THE INVENTION

The present invention relates to new methods for measuring pathogeninactivation, and in particular, new methods for measuring pathogeninactivation in blood and blood products after a decontaminationprocess. The methods involve measuring the inhibition oftemplate-dependent enzymatic synthesis of nucleic acid following theaddition of compounds that add covalently to nucleic acid, i.e.,"addition compounds". In one embodiment, the addition compounds areselected from the group comprising photoreactive compounds. In apreferred embodiment, the photoreactive compound is selected from thegroup comprising isopsoralens and psoralens.

In one embodiment, the present invention provides a method for measuringthe inactivation of pathogens in blood products, comprising thesequential steps: a) providing, in any order, i) blood productcomprising nucleic acid-containing blood cells and blood cells having nonucleic acid, ii) blood product containing means, iii) at least oneaddition compound, iv) amplification reagents, v) at least oneamplification enzyme, vi) a first and a second primer set and vii) afirst and a second amplification reaction containing means; b) adding tothe blood product in the blood product containing means an additioncompound to create a mixture; c) treating the mixture so that theaddition compound adds to the nucleic acid of the nucleic-acidcontaining blood cells; d) preparing the blood product such that thenucleic acid from the nucleic acid-containing blood cells isamplifiable; e) adding to the first amplification reaction containingmeans, in any order, a portion of the amplifiable nucleic acid, theamplification reagents, the amplification enzyme, and the first primerset, to create a reaction mixture; f) adding to the second amplificationreaction containing means, in any order, a portion of the amplifiablenucleic acid, the amplification reagents, the amplification enzyme, andthe second primer set, to create a reaction mixture; and g) exposingeach of the first and second amplification reaction containing means toconditions sufficient to provide amplification products of differentlengths.

In one embodiment the blood product comprises platelets and the nucleicacid-containing blood cells are lymphocytes.

In one embodiment, amplification is carried out using the polymerasechain reaction and a plurality of primer sets so as to provide PCRproducts of different lengths. In one embodiment, the plurality ofprimer sets are amplified together by PCR. In another embodiment, eachprimer set is amplified separately by PCR.

In one embodiment employing two different primer sets, the presentinvention contemplates that the first primer set is capable ofgenerating a product of a length short enough to be essentiallytransparent to the addition of the addition compounds to the nucleicacid under a defined set of amplification conditions (i.e., regardlessof the efficiency of the covalent addition of addition compounds, thelength of the product is sufficient such that amplification product willbe detected), and the second primer set is capable of generating aproduct of a length long enough to be affected--not completely inhibitedbut inhibited in part--by the addition of the addition compounds to thenucleic acid of the nucleic acid-containing blood cells with the sameamplification conditions as above (i.e., the efficiency of the covalentaddition of addition compounds will be reflected in the amount ofamplification product detected).

In another embodiment, the present invention provides a method formeasuring the inactivation of pathogens in blood products, comprisingthe sequential steps: a) providing, in any order, i) blood productcomprising nucleic acid-containing blood cells and blood cells having nonucleic acid, ii) blood product containing means, iii) at least oneaddition compound, iv) amplification reagents, v) at least oneamplification enzyme, vi) a first, a second and a third primer set andvii) a first, a second and a third amplification reaction containingmeans; b) adding to the blood product in the blood product containingmeans an addition compound to create a mixture; c) treating the mixtureso that the addition compound adds to the nucleic acid of thenucleic-acid containing blood cells; d) preparing the blood product suchthat the nucleic acid from the nucleic acid-containing blood cells isamplifiable; e) adding to the first amplification reaction containingmeans, in any order, a portion of the amplifiable nucleic acid, theamplification reagents, the amplification enzyme, and the first primerset, to create a reaction mixture; f) adding to the second amplificationreaction containing means, in any order, a portion of the amplifiablenucleic acid, the amplification reagents, the amplification enzyme, andthe second primer set, to create a reaction mixture; g) adding to thethird amplification reaction containing means, in any order, a portionof the amplifiable nucleic acid, the amplification reagents, theamplification enzyme, and the third primer set, to create a reactionmixture; and g) exposing each of the first, second and thirdamplification reaction containing means to conditions sufficient toprovide amplification products of different lengths.

In a preferred embodiment employing three different primer sets, thepresent invention contemplates that the first primer set is capable ofgenerating a product of a length short enough to be essentiallytransparent to the addition of the addition compounds to the nucleicacid under a defined set of amplification conditions (i.e., regardlessof the efficiency of the covalent addition of addition compounds, thelength of the product is sufficient such that amplification product willbe detected), the second primer set is capable of generating a productof a length long enough to be affected--not completely inhibited butinhibited in part--by the addition of the addition compounds to thenucleic acid of the nucleic acid-containing blood cells with the sameamplification conditions as above (i.e., the efficiency of the covalentaddition of addition compounds will be reflected in the amount ofamplification product detected), and the third primer set capable ofgenerating a product of a length long enough to be completely inhibitedby the addition of addition compounds to the nucleic acid of thenucleic-acid containing blood cells (i.e., covalent addition of theaddition compounds will be reflected by the complete absence ofmeasurable amplification product).

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the photoaddition of 8-methoxypsoralen tonormal lymphocyte DNA.

FIG. 2 is a photograph of an autoradiograph of electrophoresed,PCR-amplified, internally-radiolabelled, HLA sequences followingphotochemical decontamination of platelet concentrates containing normallymphocytes.

FIG. 3 is a graph showing the photoaddition of 8-methoxypsoralen touninfected H9 cell DNA.

FIG. 4 is a photograph of an autoradiograph of electrophoresed,PCR-amplified, internally-radiolabelled, HLA sequences followingphotochemical decontamination of platelet concentrates containinguninfected H9 cells.

FIGS. 5A and 5B graphically shows the correlation of the biologicalinactivation of HIV-1 (i.e., an RNA virus) with photoaddition of8-methoxypsoralen to viral RNA.

FIGS. 6A, B and C are photographs of an autoradiograph ofelectrophoresed, PCR-amplified, internally-radiolabelled, sequences fromthree primer pairs following photochemical decontamination of plateletconcentrates containing HIV-1 uninfected H9 cells.

DESCRIPTION OF THE INVENTION

Where decontamination of pathogens is used as a treatment for bloodproduct that will be transfused, it is essential that pathogeninactivation be complete. That is to say, partial inactivation willleave some portion of infectious organisms available to cause disease.

The present invention relates to new methods for measuring pathogeninactivation. In particular, the present invention provides new methodsfor measuring pathogen inactivation in blood and blood products afterdecontamination. The methods can be used to obtains a sensitivequantitative measurement of the efficiency of decontamination. Themethods, therefore, may be used as a quality control assay to verifyadequate treatment of blood product in any given case.

To appreciate that, in any given case, a pathogen inactivation methodmay or may not achieve complete inactivation, it is useful to consider aspecific example. A bacterial culture is said to be sterilized if analiquot of the culture, when transferred to a fresh culture plate andpermitted to grow, is undetectable after a certain time period. The timeperiod and the growth conditions (e.g., temperature) define an"amplification factor". This amplification factor along with thelimitations of the detection method (e.g., visual inspection of theculture plate for the appearance of a bacterial colony) define thesensitivity of the inactivation method. A minimal number of viablebacteria must be applied to the plate for a signal to be detectable.With the optimum detection method, this minimal number is 1 bacterialcell. With a suboptimal detection method, the minimal number ofbacterial cells applied so that a signal is observed may be much greaterthan 1. The detection method determines a "threshold" below which themethod appears to be completely effective (and above which the methodis, in fact, only partially effective).

This interplay between the amplification factor of an assay and thethreshold that the detection method defines, can be illustrated. Forexample, bacterial cells can be applied to a plate; the detection methodis arbitrarily chosen to be visual inspection. Assume the growthconditions and time are such that an overall amplification of 10⁶ hasoccurred. The detectable signal will be proportional to the number ofbacterial cells actually present after amplification. For calculationpurposes, the detection threshold is taken to be 10⁶ cells; if fewerthan 10⁶ cells are present after amplification, no cell colonies arevisually detectable and the inactivation method will appear effective.Given the amplification factor of 10⁴ and a detection threshold of 10⁶,the sensitivity limit would be 100 bacterial cells; if less than 100viable bacterial cells were present in the original aliquot of thebacterial culture after the sterilization method is performed, theculture would still appear to be sterilized.

Such a situation is common for bacterial growth assays. The sensitivityof the assay is such that viable bacterial cells are present but theassay is unable to detect them.

The same holds true for virus inactivation. In the past, inactivation ofvirus is demonstrated by a biological assay such as an enzyme assay(e.g., reduction in reverse transcriptase) or cell culture (e.g.,virus-induced host cell death). A more sensitive method has beendescribed by C. V. Hanson et al. for quantifying HIV. See J. Clin.Microbiol. 23:2030 (1990). The method is a plaque assay employingHIV-sensitive cells in a monolayer. A fluorescent stain is used anddetection is made by visualization. While the sensitivity of the assayis much improved over other methods, some small amount of viable virusmay be present even when the assay is negative. Indeed, it has beenfound that this approach to measuring viral inactivation typicallycannot distinguish the situation where inactivation of the virus iscomplete from the situation where inactivation is not complete.

Rather than measuring pathogen growth to evaluate an inactivationprocess, the methods of the present invention involve measuring theinhibition of template-dependent enzymatic synthesis of nucleic acidfollowing the addition of compounds that add covalently to nucleic acid.Enzymatic synthesis that involves nucleic acid, either solely as atemplate (e.g., translation involves the use of nucleic acid as atemplate to make polypeptides) or as both a template and a product(replication and transcription use nucleic acid as a template to producenucleic acid) is hereinafter referred to as "template-dependentenzymatic synthesis."

In the case of replication, nucleic acid polymerases replicate a nucleicacid molecule ("template") to yield a complementary ("daughter") nucleicacid molecule. For example, DNA polymerase I, isolated from E. Coli,catalyzes the addition of deoxyribonucleoside triphosphates to the 3'end of a short segment of DNA ("primer") hybridized to a template strandto yield a daughter of the template, starting from a mixture ofnucleotides (dATP, dGTP, dCTP, and dTTP). This 5' to 3'template-dependent enzymatic synthesis is also called "primerextension." The reaction will not take place in the absence of template.The reaction can be measured if one or more of the nucleotides arelabelled (usually they are radiolabelled with ³² P.

While amplification of pathogen nucleic acid is possible, it should bekept in mind that blood is subjected to decontamination on whether apathogen is present or not. Thus, when pathogen is not present, attemptsto amplify pathogen nucleic acid would result in no amplificationproduct. Secondly, the reagents for amplification of pathogen nucleicacid are typically specific for that pathogen; one would need acomprehensive method to reflect pathogen diversity. For these reasons,the preferred approach of the present invention is to amplify nucleicacid that is present in nucleic acid-containing cells of the bloodproduct rather than nucleic acid of the pathogen. This approach isgeneric; it does not rely on the specific characteristics of each andevery possible pathogen.

Normally, the amount of existing nucleic acid-containing cells in theblood product as a result of blood processing is sufficient. However,the present invention also contemplates an embodiment involving seedingthe blood product with nucleic acid for this purpose. In any event, thepresent invention contemplates that the nucleic acid may be genomic ormitochondrial DNA. Furthermore, this nucleic acid can be cellular RNA.

In one embodiment, the present invention provides a method fordetermining the efficiency of a decontamination technique (such as thePCD process) to inactivate virus in blood products (e.g., plateletconcentrates), comprising measuring the ability of the inactivationprocess to inhibit replication of intracellular nucleic acid as measuredby a nucleic acid amplification assay. In one embodiment, theamplification assay is the highly sensitive polymerase chain reaction(PCR).

PCR is a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification. See K. B. Mullis et al., U.S. Pat. Nos. 4,683,195 and4,683,202, hereby incorporated by reference. This process for amplifyingthe target sequence consists of introducing a large excess of twooligonucleotide primers to the DNA mixture containing the desired targetsequence, followed by a precise sequence of thermal cycling in thepresence of a DNA polymerase. The two primers are complementary to theirrespective strands of the double stranded target sequence. To effectamplification, the mixture is denatured and the primers then to annealedto their complementary sequences within the target molecule. Followingannealing, the primers are extended with a polymerase so as to form anew pair of complementary strands. The steps of denaturation, primerannealing, and polymerase extension can be repeated many times (i.e.,denaturation, annealing and extension constitute one "cycle;" there canbe numerous "cycles") to obtain a high concentration of an amplifiedsegment of the desired target sequence. The length of the amplifiedsegment of the desired target sequence is determined by the relativepositions of the primers with respect to each other, and therefore, thislength is a controllable parameter. By virtue of the repeating aspect ofthe process the method is referred to by the inventors as the"Polymerase Chain Reaction". Because the desired amplified segments ofthe target sequence become the predominant sequences (in terms ofconcentration) in the mixture, they are said to be "PCR amplified".

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labelled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³² P labelled deoxynucleotide triphosphates, e.g., dCTPor dATP, into the amplified sergeants). In addition to genomic DNA, anyoligonucleotide sequence can be amplified with the appropriate set ofprimer molecules.

The PCR amplification process is known to reach a plateau concentrationof specific target sequences of approximately 10⁻⁸ M. A typical reactionvolume is 100 μl, which corresponds to a yield of 6×10¹¹ double strandedproduct molecules.

"Amplification reagents" are defined as those reagents(deoxyribonucleoside triphosphates, buffers, etc.) needed foramplification except for nucleic acid, primers, and the amplificationenzyme. In one embodiment, an amplification reaction containing means isa reaction vessel (test tube, microwell, etc.).

PCR is a polynucleotide amplification protocol. The amplification factorthat is observed is related to the number (n) of cycles of PCR that haveoccurred and the efficiency of replication at each cycle (E), which inturn is a function of the priming and extension efficiencies during eachcycle. Amplification has been observed to follow the form E^(n), untilhigh concentrations of PCR product are made. At these highconcentrations (approximately 10⁻⁸ M/l) the efficiency of replicationfalls off drastically. This is probably due to the displacement of theshort oligonucleotide primers by the longer complementary strands of PCRproduct. At concentrations in excess of 10⁻⁸ M, the rate of the twocomplementary PCR amplified product strands finding each other duringthe priming reactions become sufficiently fast that this occurs beforeor concomitant with the extension step of the PCR procedure. Thisultimately leads to a reduced priming efficiency, and therefore, areduced cycle efficiency. Continued cycles of PCR lead to decliningincreases of PCR product molecules. PCR product eventually reaches aplateau concentration.

While not limited to any particular theory, the present inventioncontemplates that, when a psoralen adduct is present on nucleic acidwithin the segment of the target sequence bounded by the primersequences, the extension step of the PCR process will result in atruncated extension product that is incapable of being replicated insubsequent cycles of the PCR process.

Importantly, the inactivation will be incomplete if some of thepathogens escape modification by the decontamination process. Thisprocess is, by its nature, a statistical process. This process can becharacterized by measuring an average number (a) of adducts per DNAstrand. Not all of the strands will have a adducts per strand. If theaddition reaction is governed by Poisson statistics, the fraction ofmolecules that contain n modifications in a large population ofmolecules that have an average of a modifications is given by f_(a) (n)(see Table 1). A fraction of molecules, f_(a) (O), will contain nomodifications and are therefore considered non-sterilized, that is,"f_(a) (O)" represents the fraction of strands with zero adducts whenthe average number of adducts per strand is a and "Nf_(a) (O)"represents the number of unmodified molecules, calculated for a total of10⁶ molecules (N=10⁶).

Table 1 evaluates the unmodified fraction of DNA strands that areexpected if an average of a modifications per strand exists. Althoughthe

                  TABLE 1                                                         ______________________________________                                        f.sub.a (n) = [a.sup.n e.sup.-a ]/ n!                                         N = 10.sup.6, f.sub.a (0) = e.sup.-a                                          a            f.sub.a (0)   Nf.sub.a (0)                                       ______________________________________                                         3           0.050         5.0 × 10.sup.4                                4           0.018         1.8 × 10.sup.4                                5           0.007         6.7 × 10.sup.3                                6           0.0025        2.5 × 10.sup.3                                7           0.0009        9.1 × 10.sup.2                                8           0.0003        3.3 × 10.sup.2                                9           0.00012       1.2 × 10.sup.2                               10           0.000045      4.5 × 10.sup.1                               11           0.000017      17.0                                               12           0.0000061     6.1                                                13           0.0000023     2.2                                                14           0.00000083    .8                                                 15           0.00000030    .3                                                 16           0.00000011    .1                                                 17           0.00000004    0.04                                               ______________________________________                                    

fraction of molecules with no modifications is small for all values ofa, the expected number of unmodified molecules is large when the PCDprocess is applied to a large number of molecules (N). For example,Table 1 shows that 2.5=10³ unmodified molecules are expected if there isan average of 6 effective adducts per strand in a population of 10⁶strands. Effective adducts are those adducts that occur in the segmentof a target molecule that is bounded by the primer sequences. Wherephotoreactive compounds are employed, alterations of the modificationdensity can be expected through the use of different photoreactivecompounds, or the use of the same photoreactive compound at differentconcentrations. In particular, the modification density is expected toincrease through the use of the same photochemical agent at higherconcentrations or the use of longer irradiation times.

For a fixed modification density the present invention contemplatesanother method of improving the sensitivity limit for evaluating the PCDprocess. The important statistical parameter is the average number ofadducts per PCR strand. By choosing PCR primers judiciously, the lengthof the PCR products can be varied, and therefore, the average number ofadducts per strand can be varied. The present invention, therefore,contemplates the use of a plurality of primer sets so as to provide PCRproducts of different lengths.

In one embodiment, the present invention contemplates two differentprimer sets: the first generating a product of a length short enough tobe essentially transparent to the PCD process (i.e., regardless of theefficiency of the covalent addition of photoreactive compounds, PCRproduct will be detected under a defined set of amplificationconditions), and the second generating a product of a length long enoughto be affected--but not completely inhibited--by the PCD process (i.e.,the efficiency of the covalent addition of photoreactive compounds willbe reflected in the amount of PCR product detected).

In a preferred embodiment, the present invention contemplates threedifferent primer sets: the first generating a product of a length shortenough to be essentially transparent to the PCD process, the secondgenerating a product of a length long enough to be affected--but notcompletely inhibited--by the PCD process, and the third generating aproduct of a length long enough to be completely inhibited by the PCDprocess (i.e., covalent addition of photoreactive compounds will bereflected by the complete absence of measurable PCR product).

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); gm (grams); mg (milligrams); μg (micrograms); L (liters);ml (milliliters); μ(microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); °C. (degrees Centigrade); Ci (Curies);MW (molecular weight); OD (optical density); DMSO (dimethyl sulfoxide);EDTA (ethylenediamine-tetracetic acid); 1xTE (buffer: 10 mM Tris/1 mMEDTA, pH 7.5); 1xTaq (buffer: 50 mM KCl, 2.5 mM MgCl₂, 10 mM Tris, pH8.5, 200 μg/ml gelatin); PAGE (polyacrylamide gel electrophoresis); UV(ultraviolet); V (volts); W (watts); mA (milliamps); bp (base pair); CPM(counts per minute).

In many of the examples below, 8-methoxypsoralen is used. Generally,stock 8-MOP is diluted in DMSO (100 mg/ml) and added to a finalconcentration of 300 μg/ml.

In some instances below, amplification of human histocompatibility (HLA)Class II genes was performed using primer pair GH26/GH27 and humanplacental DNA to produce a 242-mer product. The sequences of theseprimers are:

    __________________________________________________________________________    GH26                                                                              5'-GTGCTGCAGGTGTAAACTTGTACCAG-3'                                                                        (Seq. Id. No. 1)                                GH27                                                                              5'-CACGGATCCGGTAGCAGCGGTAGAGTTG-3'                                                                      (Seq. Id. No. 2)                                __________________________________________________________________________

These primers and other primers are described in PCR Protocols: A GuideTo Methods and Applications, Innis, M. A. et al. eds., pp. 261-271(1990)).

In another case, amplification of human globin sequences was performedusing primer pair KM38/RS118 to produce a 135-mer product and RS40/RS80to produce a 989-met product. The sequences of these primers are:

    __________________________________________________________________________    KM-38 5'-TGGTCTCCTTAAACCTGTCTTG-3'                                                                        (Seq. Id. No. 3)                                  RS-118                                                                              5'-ACACCATGGTGCACCTGACT-3'                                                                          (Seq. Id. No. 4)                                  RS-40 5'-ATTTTCCCACCCTTAGGCTG-3'                                                                          (Seq. Id. No. 5)                                  RS-80 5'-TGGTAGCTGGATTGTAGCTC-3'                                                                          (Seq. Id. No. 6)                                  __________________________________________________________________________

Denaturing (8M urea) polyacrylamide mini-gels were used. 10 to 12.5%gels were used for oligonucleotides between 40 and 400 base pairs inlength; 6 to 8% gels were used for longer sequences. Depending on thelength of DNA to be analyzed, samples were loaded in either 8M urea,containing 0,025% tracking dyes (bromphenol blue and xylene cyanol), orin 80% formamide, 10% glycerol, 0.025% tracking dyes, thenelectrophoresed for 30-60 minutes at 300 Volts. Following PAGE,individual bands were, in most cases, visualized by autoradiography.Autoradiography involved exposure overnight at -70° C. to Kodak XAR-5films with an intensifying screen.

In order to visualize with autoradiography, PCR products were internallyradiolabelled. This simply involved adding 2 μCi of α-³² P-dCTP (3000Ci/mmole, NEN Research Products, Boston Mass., U.S.A.) to each PCRreaction. The internally-radiolabelled PCR products are directlyfractionated by this denaturing PAGE.

In some cases, the visualized bands were cut from the gel and collectedfor scintillation counting. Scintillation counting involved the use of ascintillation fluid and a commercial scintillation counter (SearleAnalytic 92, Model #000 006893).

Generally, PCR was carried out using 175-200 μM dNTPs(deoxyribonucleoside 5'-triphosphates) and 0.1 to 1.0 μM primers. 2.5 to5.0 Units/ 100 μl of Taq polymerase was used. PCR reactions wereoverlaid with 30-100 μl light mineral oil. A typical PCR cycle for HIVamplification using a Perkin-Elmer Cetus DNA Thermal Cycler (Part No.N8010150) was: denaturation at 95° C. for 30 seconds; annealing at 55°C. for 30 seconds; and extension at 72° C. for 1 minute. PCR cycles werenormally carried out in this manner for between 20 to 30 cycles followedby 7 minutes at 72° C. PCR product or "amplicon" was then detected.

In some of the examples below, platelet concentrates are used. Plateletsare prepared by differential centrifugation of whole blood collectedfrom random blood donors. The preparation step concentrates theplatelets in reduced volumes of plasma ranging from about 50 to 100 ml.The concentrates contain approximately 5.5×10¹⁰ platelets per bag aswell as a variable amount (10⁷ to 10⁸) of contaminating lymphocytes.

Unless otherwise indicated, the UVA irradiation device consists of twobanks of Sylvania Dermacontrol lamps (one above, and one below, thesample) capable of producing approximately 20 mW/cm of light between320-400 nm. The approximate irradiation path length of the plateletmixture in the irradiation chamber was 0.5 cm.

EXAMPLE 1

In this experiment, the amount of 8-MOP bound to normal lymphocytesseeded into platelets and Factor VIII concentrates was measured. Asstarting material, 0.8×10⁸ lymphocytes were seeded into a unit ofplatelet concentrates or 10 ml of clotting Factor VIII concentrates.8-MOP was added to a final concentration of 300 ug/ml. ³ H-8-MOP wasadded to a specific activity of 4.73 uCi/mg. The platelets wereirradiated in their storage bag (PL 732, Fenwal, Baxter). The FactorVIII concentrate was irradiated in a petri dish. The samples wereirradiated from above and below by GE type F20T12-BLB fluorescent UVA(320-400 nm) bulbs with an electric fan blowing gently across the lightsto cool the area. The temperature was maintained between 22° and 27° C.during irradiation. The platelet irradiation was carried out in anatmosphere of 5% CO₂ and 95% N₂. Available UVA light was measured (by aBlack-ray longwave UV meter) with both light banks turned on. The totalmeasured light intensity was 3.5-4.8 mW/cm². The Factor VIII concentrateirradiation was carried out with no O₂ control. Control samples includedone which received no treatment and one to which only 8-MOP was addedand no UVA light was applied.

Following irradiation, total cellular lymphocyte DNA was extracted byphenol extraction following proteinase K digestion. The DNA was thenethanol precipitated three times. The number of 8-MOP moleculescovalently bound per 1,000 base pair was determined by measuring thequantity of DNA (A₂₆₀ nm) using a spectrophotometer and the ³ H-CPMassociated with the DNA using a scintillation counter. FIG. 1 shows that8-MOP adduct formation is a function of UVA irradiation time. Treatmentwith 8-MOP alone without UVA light for up to 10 hours resulted in no8-MOP addition to cellular DNA. For UVA irradiated plateletconcentrates, the number of 8-MOP adducts increased with time to a levelof 17.2 adducts/1,000 bp. For Factor VIII concentrates, 8-MOP adductsreached a level of 43.5/1,000 bp after 6 hours of UVA irradiation.

EXAMPLE 2

In this experiment, the ability of PCD treatment to inhibit replicationof a small segment of cellular DNA was determined. The nucleic acid fromExample 1 was processed using the PCR reaction to determine if a smallsegment of cellular DNA could be replicated. Lymphocyte DNA (1 ug oftotal DNA) was utilized as the template with the human HLA primers, GH26and GH27 (Cetus Corp., Emeryville, Calif.). In these experiments the PCRreaction was run for 20 cycles. The PCR products were analyzed byelectrophoresis on a 12% denaturing polyacrylamide gel andautoradiography (FIG. 2).

The predominant PCR product of untreated lymphocyte DNA using theseprimers (FIG. 2, lane 6) is a 242 bp fragment in the HLA-DQA locus. Thepresence of 10 μg/ml 8-MOP (FIG. 2, lane 7) or 2% DMSO (FIG. 2, lane 8)did not inhibit the PCR reactions. A similar amount of the 242 bpfragment was detected using lymphocyte DNA treated with 8-MOP withoutUVA (FIG. 2, lane 1).

In contrast, PCR mediated replication of this DNA segment was markedlyinhibited when PCD treated lymphocyte DNA was used as the template (FIG.2, lanes 2-5). The degree of inhibition appears to be a function of thenumber of 8-MOP adducts within each template. Only a small amount of the242 bp fragment was detected if 8-MOP adducts were present at an averageof 5.6 per 1,000 bp (FIG. 2, lane 2), none of the 242 bp fragment wasvisible in samples where 8-MOP adducts were present at levels greaterthan 12 per 1,000 bp (FIG. 2, lanes 3, 4 and 5). Thus, at levels of8-MOP addition achievable in lymphocytes contaminating blood products,PCR mediated replication of a very small DNA segment appears to bestrongly inhibited. This 242 bp DNA segment codes for 80 amino acidswhich is far smaller than any potentially infectious provital DNAsegment. Furthermore, the reagents used in the PCD technique do notinterfere with the PCR assay system. This experiment indicates thatpsoralen adducts block replication of genomic DNA of lymphocytes inblood products. It also is highly suggestive that this method will beeffective against intranuclear proviral DNA.

EXAMPLE 3

Example 1 demonstrated 8-MOP photoaddition to normal lymphocyte DNA inplatelet concentrates and Example 2 showed that this modification couldbe detected by PCR, consistent with data published earlier (posterpresentation at the 31st annual meeting of The American Society ofHematology, 1989). In this example, the photoaddition kinetics of 8-MOPto cellular DNA of H9 cells added to platelet concentrates areestablished.

Uninfected (2.5×10⁷) H9 cells in one ml of Dulbecco's modified Eagle'sminimum essential medium (DMEM)/15% fetal bovine serum (FBS) were addedto one unit of platelet concentrate. Stock 8-MOP (100 mg/ml in DMSO) wasadded to a final concentration of 300 μg/ml. ³ H-8-MOP was added to aspecific activity of 118 mCi/mmole. 5 ml of the mixture was transferredto a siliconized glass chamber equipped with an outer jacket in which H₂O was circulated to maintain the temperature at 25° C. The gas phaseabove the platelet suspension was flushed with 95% N₂ /5% Co₂ for 30minutes in order to reduce the concentration of O₂ in the plateletconcentrate and then tightly capped before placing in the Dermacontrolirradiation device (described above). Individual samples in separatechambers were set up for each UVA irradiation time point. The controlsamples included an untreated sample and a sample to which only 8-MOPwas added.

H9 cells were recovered from the platelet concentrate by centrifugation.The total DNA was purified using the standard phenol/chloroformextraction procedure following proteinase K digestion. The DNA contentwas determined spectrophotometrically after 2 and 3 ethanolprecipitations. The ³ H-8-MOP content in the same sample used for thespectrophotometric measurement was determined by liquid scintillationcounting. The level of DNA photomodification was calculated from thespecific activity of the added ³ H-8-MOP.

As shown in FIG. 3, the extent of 8-MOP photomodification of cellularDNA was a function of the time of UVA irradiation. The 8-MOP addition tocellular DNA was UVA dependent since no 8-MOP adducts were formed after4 hours of incubation in the dark.

The same DNA samples prepared for the determination of 8-MOP photoadductlevels as described above were used for the PCR analysis. One μg(containing 3×10⁵ copies of the HLA-DQα sequence) of each DNApreparation was used per 100 μl of a PCR reaction. Each PCR reactioncontains in 100 μl: 10 mM Tris-buffer pH 8.4, 50 mM KCl, 2.5 mM MgCl₂,0.02% gelatin, 200 μM each of dATP, dCTP, dGTP and dTTP, 20 μCi of α⁻³²P-dCTP (3000 Ci/mmole), 50 pmoles of the upstream primer GH26, 50 pmolesof the downstream primer GH27 and 2.5 units of Taq polymerase. The DNAwas amplified using the thermal profile: 95° C. for 30", 55° C. fir 30",and 72° C. for 1'. Samples of 20 μl each were withdrawn for analysisafter 20, 25 and 30 cycles of PCR. The amplified products were separatedfrom the unincorporated α-³² P-dCTP by polyacrylamide gelelectrophoresis (PAGE) through a 12.5% denaturing gel containing 8M ureaand visualized by autoradiography.

The results (FIG. 4) indicate that 8-MOP photoadducts on H9 cellular DNAinhibit amplification of HLA sequences. The extent of inhibition is afunction of the number of adducts formed. The amplification reactionproceeded normally with the untreated DNA sample (lane 2) and the 4 hour8-MOP (no UVA) treated DNA sample (lane 3), since neither of thesesamples contained 8-MOP photoadducts. By contrast, the level ofamplification was drastically reduced in samples containing 8-MOPphotomodification of the DNA (lanes 4-7). No amplification was presentafter 20 cycles when greater than 8 adducts per 1,000 bp were formed. At25 cycles, the intensity gradient of amplified products correlated withthe number of 8-MOP adducts on the DNA. At 30 cycles the PCR inhibitionis still clearly evident.

Bands were excised from the gel and radioactivity was determined byscintillation counting. The amplification efficiency expressed as therelative PCR signal for 25 and 30 PCR cycles was determined from theratio of the radioactivity of the amplicon bands (Table 2). It is clearthat higher levels of photomodification resulted in a higher degree ofPCR inhibition. The amplification efficiency was not determined for the20 cycle-PCR because no amplicon bands were detected for the modifiedDNA.

                  TABLE 2                                                         ______________________________________                                        Comparison of the Relative PCR                                                Amplification Signal of 8-MOP/UVA Treated                                     Cellular DNA With Untreated Control DNA.                                      Sam-            UVA     8-MOP per                                                                             Relative PCR Signal                           ple  Treatment  (hr)    1,000 bp                                                                              25X     30X                                   ______________________________________                                        1    Reagent Only                                                                             0       0       0       0                                     2    Control    0       0       100     100                                   3    8-MOP only 0       0       100     100                                   4    8-MOP/UVA  4       22      0.34    2.3                                   5    8-MOP/UVA  2       17      0.51    3.2                                   6    8-MOP/UVA  1       15      0.84    5.1                                   7    8-MOP/UVA    0.5   8       1.76    10.8                                  ______________________________________                                    

At a level greater than 8 adducts/1,000 bp, no amplification product wasdetected after 20 cycles of PCR. After 25 cycles of PCR, theamplification product of modified DNA was detected at the level of0.34-1.76% Of the control unmodified cellular DNA. The 1.76% level ofdetection corresponds to the 0.5 hour UVA irradiation, at which point2.7 logs of HIV-1 was inactivated (see FIG. 5 and accompanyingdiscussion below). While 0.84% amplification was detected after 1 hourof irradiation, the HIV-1 activity was undetectable by the microplaqueassay (FIG. 5). These results indicate that PCR is a surrogate testwhich can measure the extent of inactivating modifications with moresensitivity than the biological assays. The use of increased number ofPCR cycles permits quantitation of the effects of the inactivationtreatment over a range where such effects may only be inferred byextrapolation when infectivity data alone are used.

EXAMPLE 4

Having established the photoaddition kinetics of 8-MOP to cellular DNAof uninfected H9 cells, this experiment establishes the inactivationkinetics of HIV-1 using infected H9 cells. Infected (2.5×10⁷) H9 cellsin one ml of Dulbecco's modified Eagle's minimum essential medium(DMEM)/15% fetal bovine serum (FBS) were added to one unit of plateletconcentrate. Stock 8-MOP (100 mg/ml in DMSO) was added to a finalconcentration of 300 μg/ml. 5 ml of the mixture was transferred to asiliconized glass chamber equipped with an outer jacket in which H₂ Owas circulated to maintain the temperature at 25° C. The gas phase abovethe platelet suspension was flushed with 95% N.sub. /5% Co₂ for 30minutes in order to reduce the concentration of O₂ in the plateletconcentrate and then tightly capped before placing in the irradiationdevice (described above). Individual samples in separate chambers wereset up for each UVA irradiation time point. The control samples includedan untreated sample and a sample to which only 8-MOP was added. TheHIV-1 infectivity for each sample was measuring using the microplaqueassay described by C. V. Hanson et al. for quantitating HIV. See J.Clin. Microbiol. 23:2030 (1990).

As seen in FIG. 5A, HIV-1 was inactivated in platelet concentrates at arate of 4 to 5 logs/hr under the 8-MOP/UVA treatment conditions used.The viral inactivation was clearly due to the photochemical treatmentsince no HIV-1 inactivation was detected if either 8-MOP or UVA was usedalone. After one or more hours of 8-MOP/UVA treatment, HIV activity wasbelow the level of detectability by the plaque assay (indicated by thearrows).

In a parallel experiment (FIG. 5B), samples were irradiated for shortertime periods. A log-linear inactivation rate was obtained.

The experimental limitations do not allow measurements of greater than 4logs of titer loss, which occurs within the first 60 minutes of exposureto 8-MOP. The important question is how to extrapolate further at thelonger time points. The DNA binding data show that adducts arecontinually being formed out to at least 4 hrs (FIG. 3). How does theDNA binding data correlate with viral inactivation and how should it beused to estimate killing in the inactivation region for which directmeasurements are not available? It should be kept in mind that thegenomic targets are completely different (double-stranded DNA vs.encapsulated RNA in viral particles). One would expect that the bindingby 8-MOP (which strongly prefers a double-stranded helical region) willbe substantially less with the viral RNA target compared with nuclearDNA.

It is possible to relate the DNA data to RNA modifications by making theassumption that a single adduct per infectious RNA molecule/virusparticle is sufficient to inactivate virus. With this assumption, thenumber of RNA adducts can be indirectly calculated from the inactivationdata via Poisson statistics:

Inactivated fraction=10^(-log) survival =e^(-a),

where a equals the average number of adducts per infectious HIV RNAmolecule.

Based upon the experimental data, the range of adducts per infectiousRNA molecule is 2.07 adducts at 5 minutes to 9.2 adducts at the 1 hourtime point. This data is shown in FIG. 5B. The comparative data (FIG. 3versus FIG. 5B) verifies expectations that RNA binding is substantiallyless than the DNA binding. 9.2 adducts per RNA genome (10,000 bases) maybe equivalent to 18.4 adducts per 10,000 base pairs or 1.84 adducts per1,000 bp. At the equivalent time point the DNA binding data yields 14DNA adducts per 1,000 bp's, corresponding to a 7.6 fold difference inrelative reactivity.

If one assumes that the differential DNA/RNA reactivity is constant overtime, one can calculate the log of survival at the longer time pointsfrom the DNA data. At 4 hours, there are 22 DNA adducts. Using the 7.6fold difference in relative reactivity, this corresponds to 14.5 adductsper RNA genome. At this average modification density per RNA genome, thefrequency of an HIV RNA genome without a single 8-MOP adduct is e⁻¹⁴.5=5×10⁻⁷. Seven logs of inactivation is well beyond the measurementcapabilities of current HIV biological assays.

To consider whether the PCR data provides a good check for themodification numbers via a "biological" measurement, one may use theexperimentally obtained number of 90,000 CPM at 25 cycles ofamplification of the HLA amplification of the 242mer vs. 300 CPM for the8-MOP, 4 hour-irradiated samples (22 adducts per 1,000 bp's by ³ Hcounts). From the above data and assuming that the PCR did not plateau,there is a 300 fold inactivation of this PCR sequence. The averagenumber of adducts per amplification unit to account for thisinactivation is related as:

1/300=e^(-a), which yields an average of 5.7 adducts per 242-mer.

This average number of adducts from the PCR data is extremely close tothat measured by the tritium incorporation assay:

5.7 adducts/242 base pairs=23.5 adducts per 1,000 bp's.

Thus, the relative reduction in PCR signal corresponds to the level of8-MOP addition to cellular DNA.

EXAMPLE 5

Having established the inactivation kinetics of HIV-1, it is nowpossible to demonstrate the method of the present invention formeasuring inactivation. A preferred embodiment of the present inventioncontemplates employing three different primer sets in conjunction withthe PCD process: the first generating a product of a length short enoughto be essentially transparent to the PCD process, the second generatinga product of a length long enough to be affected--but not completelyinhibited--by the PCD process, and the third generating a product of alength long enough to be completely inhibited by the PCD process (i.e.,covalent addition of photoreactive compounds will be reflected by thecomplete absence of measurable PCR product).

Uninfected (2.5×10⁷) H9 cells in one ml of Dulbecco's modified Eagle'sminimum essential medium (DMEM)/15% fetal bovine serum (FBS) were addedto one unit of platelet concentrate. Stock 8-MOP (100 mg/ml in DMSO) wasadded to a final concentration of 300 μg/ml. ₃ H-8-MOP was added to aspecific activity of 118 mCi/mmole. 5 ml of the mixture was transferredto a siliconized glass chamber equipped with an outer jacket in which H₂O was circulated to maintain the temperature at 25° C. The gas phaseabove the platelet suspension was flushed with 95% N₂ /5% CO₂ for 30minutes in order to reduce the concentration of O₂ in the plateletconcentrate and then tightly capped before placing in the irradiationdevice (described above). Individual samples in separate chambers wereset up for each UVA irradiation time point. The control samples includedan untreated sample and a sample to which only 8-MOP was added.

H9 cells were recovered from the platelet concentrate by centrifugation.The total DNA was purified using the standard phenol/chloroformextraction procedure following proteinase K digestion. Samples wereprepared for PCR. One μg (containing 3×10⁵ copies of the HLA-DQαsequence) of each DNA preparation was used per 100 μl of a PCR reaction.Each PCR reaction contains in 100 μl: 10 mM Tris-buffer pH 8.4, 50 mMKCl, 2.5 mM MgCl₂, 0.02% gelatin, 200 μM each of dATP, dCTP, dGTP anddTTP, 20 μCi of α⁻³² P-dCTP (3000 Ci/mmole), 50 pmoles of the upstreamprimer, and 50 pmoles of the downstream primer and 2.5 units of Taqpolymerase.

Three primer sets were used: RS40/RS80, GH26/GH27 and KM38/RS118. Theamplified products were separated from the unincorporated α⁻⁼ P-dCTP byelectrophoresis through denaturing PAGE with the percentage ofpolyacrylimide specific for each amplicon:

6% gel for RS40/RS80 (989 bp amplicon)

10% gel for GH26/GH27 (242 bp amplicon)

12.5% gel for KM38/RS118 (135 bp amplicon)

The results were visualized by autoradiography (FIG. 6). Using primersets that are far from each other, e.g., RS40/RS80 for a 989 bpamplicon, there are enough 8-MOP adducts to "completely" inhibit PCReven after 30 cycles. Therefore, they can be used as an internalnegative control (FIG. 6A). Using primer sets that are very close toeach other, e.g., HM38/RS118 for the 135 bp amplicon, there will not beenough 8-MOP adducts to inhibit PCR (i.e., it is transparent tophotoaddition), therefore they can be used as an internal positivecontrol (FIG. 6C). Most importantly, with the HLA DQα primer set (FIG.6B), a high degree of PCR inhibition is evident, demonstrating theefficacy of the PCD treatment.

For quantitation, the visualized bands were cut from the gels andcollected for scintillation counting. Tables 3-5 correspond to FIGS. 6A,6B and 6C, respectively. The raw CPM are shown for band in each gellane. The number of PCR cycles is also indicated with the adducts per1,000 base pair indicated in parenthesis. The Normalized Value is alsogiven.

                  TABLE 3                                                         ______________________________________                                        Relative PCR Amplification Signal from                                        primer pair RS40/RS80.                                                        Lane    Treatment   CPM/band  Normalized Value                                ______________________________________                                        1       Neg. Control                                                                              316       --                                              2       20X (0)     4,766     --                                              3       20X (22)    236       --                                              4       20X (8)     440       --                                              5       25X (0)     15,125    100                                             6       25X (22)    558       1.6                                             7       25X (8)     526       1.4                                             8       30X (0)     31,294    100                                             9       30X (22)    494       0.6                                             10      30X (8)     686       1.2                                             ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Relative PCR Amplification Signal from                                        primer pair GH26/GH27.                                                        Lane    Treatment   CPM/band  Normalized Value                                ______________________________________                                        1       Neg. Control                                                                              252       --                                              2       20X (0)     1,286     --                                              3       20X (22)    270       --                                              4       20X (8)     294       --                                              5       25X (0)     9,510     100                                             6       25X (22)    270       0.2                                             7       25X (8)     458       2.2                                             8       30X (0)     37,252    100                                             9       30X (22)    1,010     2.0                                             10      30X (8)     3,682     9.3                                             ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Relative PCR Amplification Signal from                                        primer pair KM38/RS118.                                                       Lane    Treatment   CPM/band  Normalized Value                                ______________________________________                                        1       Neg. Control                                                                                339     --                                              2       20X (0)     2,768     --                                              3       20X (22)      468     --                                              4       20X (8)       532     --                                              5       25X (0)     35,372    100                                             6       25X (22)    1,026     2.0                                             7       25X (8)     1,828     4.3                                             8       30X (0)     101,046   100                                             9       30X (22)    8,158     7.8                                             10      30X (8)     22,514    22.0                                            ______________________________________                                    

The Normalized Value is calculated using the raw CPM. For each cyclenumber, the CPM obtained using 8-MOP modified nucleic acid are dividedby the CPM achieved in the case where unmodified nucleic acid is used.The latter case is taken at the value 100. The values at 20 cycles werenot calculated, given the low raw CPM.

It is clear that the higher modification density results in fewer CPMand, consequently a lower normalized value. Thus, the normalized valuecan serve as a quantitative method for measuring pathogen inactivation.

EXAMPLE 6

While the present invention contemplates an embodiment involving seedingthe blood product with nucleic acid, normally the amount of existingnucleic acid-containing cells in the blood product as a result of bloodprocessing is sufficient. For this purpose, it is desirable to isolatenucleic acid from contaminating lymphocytes in the blood product.

8-MOP is added to one unit of platelet concentrate. 5 ml of the mixtureis transferred to a siliconized glass chamber equipped with an outerjacket in which H₂ O is circulated to maintain the temperature at 25° C.The gas phase above the platelet suspension is flushed with 95% N₂ /5%CO₂ for 30 minutes in order to reduce the concentration of O₂ in theplatelet concentrate and then tightly capped before placing in theirradiation device (described above). Individual samples in separatechambers are set up for each UVA irradiation time point. The controlsamples included an untreated sample and a sample to which only 8-MOPwas added.

Nucleic acid from lymphocytes is prepared from the platelet concentrateby centrifuging to create a white cell pellet and a supernatant. Thesupernatant is removed and the pellet is resuspended in ISOTON®II(Coulter Diagnostics, a division of Coulter Electronics, Inc., Hialeah,Fla., U.S.A.). Following a second centrifugation and wash, the cells arepelleted and thereafter lysed by the addition of protease K (50° C. for1 hour). The protease is inactivated by heating at 95° C.

Aliquots of the lysate are added to standard PCR reactions and amplifiedwith the three primer sets as in Example 5, above. The results arevisualized by autoradiography to demonstrate the efficacy of the PCDtreatment.

EXAMPLE 7

The assay is performed as in Example 6 except that the isopsoralen4'-aminomethyl-4,5'-dimethylisopsoralen (AMDMIP) is substituted for thepsoralen 8-MOP. AMDMIP is commercially available or can be synthesizedaccording to the method of Baccichetti et al., U.S. Pat. No. 4,312,883,hereby incorporated by reference.

EXAMPLE 8

As noted previously, normally, the amount of existing nucleicacid-containing cells in the blood product as a result of bloodprocessing is sufficient to measure inactivation of pathogens in bloodproduct. Furthermore, the present invention contemplates that thenucleic acid may be mitochondrial DNA ("mtDNA") such as that found inplatelets.

Because mtDNA differences among animal species are large, primers can beselected that amplify specific segments of human mtDNA. On the otherhand, primers can be selected that amplify the corresponding segments ofmtDNA from other species. See Kocher et al., Proc. Natl. Acad. Sci. USA86:6196 (1989). The sequences of the seven primers follow, the letters Land H refer to the light and heavy strands, and the number refers to theposition of the 3' base of the primer in the complete human mtDNAsequence:

    __________________________________________________________________________    L14841                                                                              (5'-AAAAAGCTTCCATCCAACATCTCAGCATGATGAAA-3')                                                                   (Seq. Id. No. 7)                        and H15149                                                                          (5'-AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-3')                                                                    (Seq. Id. No. 8)                                                              (cytochrome b);                         L1091 (5'-AAAAAGCTTCAAACTGGGATTAGATACCCCACTAT-3')                                                                   (Seq. Id. No. 9)                        and H1478                                                                           (5'-TGACTGCAGAGGGTGACGGGCGGTGTGT-3')                                                                          (Seq. Id. No. 10)                                                             (12S rRNA);                             L15926                                                                              (5'-TCAAAGCTTACACCAGTCTTGTAAACC-3'),                                                                          (Seq. Id. No. 11)                       L16007                                                                              (5'-CCCAAAGCTAAAATTCTAA-3'),    (Seq. Id. No. 12)                       and H00651                                                                          (5'-TAACTGCAGAAGGCTAGGACCAAACCT-3')                                                                           (Seq. Id. No. 13)                                                             (control region).                       __________________________________________________________________________

The preferred primers are the last three primers above. These primersare expected not to amplify pathogen sequences.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 13                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTGCTGCAGGTGTAAACTTGTACCAG26                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (v ) FRAGMENT TYPE: internal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CACGGATCCGGTAGCAGCGGTAGAGTTG28                                                (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (i i) MOLECULE TYPE: DNA (genomic)                                            (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TGGTCTCCTTAAACCTGTCTTG22                                                      (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ACACCATGGTGCACCTGACT20                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ATTTTCCCACCCTTAGGCTG20                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     ( B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TGGTAGCTGGATTGTAGCTG20                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AAAAAGCTTCCATCCAACATCTCAGCATGATGAAA35                                         (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA34                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AAAAAGCTTCAAACTGGGATTAGATACCCCACTAT 35                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TGACTGCAGAGGGTGACGGGCGGTGTGT 28                                               (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TCAAAGCTTACACCAGTCTTGTAAACC 27                                                (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CCCAAAGCTAAAATTC TAA19                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      T AACTGCAGAAGGCTAGGACCAAACCT27                                            

We claim:
 1. A method for measuring the inactivation of pathogens inblood products, comprising the sequential steps:a) providing i) a bloodproduct comprising nucleic acid-containing blood cells and blood cellshaving no nucleic acid, ii) blood product containing means, iii) atleast one photoreactive compound selected from the group consisting ofpsoralens, iv) amplification reagents, v) at least one amplificationenzyme, vi) a first and a second primer set vii) a first and a secondamplification reaction containing means, and viii.) a photoactivationdevice capable of emitting a given intensity of a spectrum ofelectromagnetic radiation comprising wavelengths of approximately 320 nmand greater; b) adding to said blood product in said blood productcontaining means said photoreactive compound to create a mixture; c)treating said mixture in said photoactivation device so that saidphotoreactive compound adds to said nucleic acid of said nucleic-acidcontaining blood cells; d) preparing said blood product such that saidnucleic acid from said nucleic acid-containing blood cells isamplifiable nucleic acid; e) adding to said first amplification reactioncontaining means, in any order, a portion of said amplifiable nucleicacid, said amplification reagents, said amplification enzyme, and saidfirst primer set, to create a reaction mixture; f) adding to said secondamplification reaction containing means, in any order, a portion of saidamplifiable nucleic acid, said amplification reagents, saidamplification enzyme, and said second primer set, to create a reactionmixture; g) exposing each of said first and second amplificationreaction containing means to conditions sufficient such that said firstprimer set generates a product in said amplification reaction of alength sufficient to be essentially uninhibited by the addition of saidphotoreactive compounds to said nucleic acid of said nucleic-acidcontaining blood cell, and such that said second primer set generates aproduct in said amplification reaction of a length sufficient to beinhibited in part by the addition of said photoreactive compound to saidnucleic acid of said nucleic-acid containing blood cell; and h)comparing the quantity of said amplification products in said first andsecond amplification reaction containing means.
 2. The method of claim 1wherein said blood product comprises platelets.
 3. The method of claim 1wherein said nucleic acid-containing blood cells are lymphocytes.
 4. Themethod of claim 1 wherein said amplification reagents comprisePolymerase chain reaction reagents.
 5. The method of claim 1 whereinsaid amplification enzyme is a thermally stable polymerase.
 6. A methodfor measuring the inactivation of pathogens in blood products,comprising the sequential steps:a) providing i) a blood productcomprising nucleic acid-containing blood cells and blood cells having nonucleic acid, ii) blood product containing means, iii) at least onephotoreactive compound selected from the group consisting of psoralens,iv) amplification reagents, v) at least one amplification enzyme, vi) afirst, a second and a third primer set vii) a first, a second and athird amplification reaction containing means, and viii) aphotoactivation device capable of emitting a given intensity of aspectrum of electromagnetic radiation comprising wavelengths ofapproximately 320 nm and greater; b) adding to said blood product insaid blood product containing means said photoreactive compound tocreate a mixture; c) treating said mixture in said photoactivationdevice so that said photoreactive compound adds to said nucleic acid ofsaid nucleic-acid containing blood cells; d) preparing said bloodproduct such that said nucleic acid from said nucleic acid-containingblood cells is amplifiable nucleic acid; e) adding to said firstamplification reaction containing means, in any order, a portion of saidamplifiable nucleic acid, said amplification reagents, saidamplification enzyme, and said first primer set, to create a reactionmixture; f) adding to said second amplification reaction containingmeans, in any order, a portion of said amplifiable nucleic acid, saidamplification reagents, said amplification enzyme, and said secondprimer set, to create a reaction mixture; g) adding to said thirdamplification reaction containing means, in any order, a portion of saidamplifiable nucleic acid, said amplification reagents, saidamplification enzyme, and said third primer set, to create a reactionmixture; h) exposing each of said first, second and third amplificationreaction containing means to conditions sufficient such that said firstprimer set generates a product in said amplification reaction of alength sufficient to be essentially uninhibited by the addition of saidphotoreactive compounds to said nucleic acid of said nucleic-acidcontaining blood cell, such that said second primer set generates aproduct in said amplification reaction of a length sufficient to beinhibited in part by the addition of said photoreactive compounds tosaid nucleic acid of said nucleic-acid containing blood cell, and suchthat said third primer set generates a product in said amplificationreaction of a length sufficient to be completely inhibited by theaddition of said photoreactive compounds to said nucleic acid of saidnucleic-acid containing blood cell; and i) comparing the quantity ofsaid amplification products in said first, second and thirdamplification reaction containing means.
 7. The method of claim 6wherein said blood product comprises platelets.
 8. The method of claim 6wherein said nucleic acid-containing blood cells are lymphocytes.
 9. Themethod of claim 6 wherein said amplification reagents comprisePolymerase chain reaction reagents.
 10. The method of claim 6 whereinsaid amplification enzyme is a thermally stable polymerase.
 11. A methodof estimating inactivation of one or more pathogens by measuring thephotomodification of endogenous nucleic acid in a blood product havingblood cells that contain nucleic acid, comprising the steps:a) adding apsoralen which, upon photoactivation with a spectrum of electromagneticradiation comprising wavelengths of approximately 320 nm and greater,binds covalently with nucleic acid to said blood product to photomodifyat least some of said endogenous nucleic acid; b) treating saidendogenous nucleic acid from said blood product to make it accessible toamplification reagents; c) adding a first portion of said endogenousnucleic acid of step (b) to a first amplification reaction containingmeans in the presence of a first primer set which can amplify a firsttarget sequence if said first target sequence is unmodified; d) adding asecond portion of said endogenous nucleic acid of step (b) to a secondamplification reaction containing means in the presence of a secondprimer set which can amplify a second target sequence havingsufficiently fewer base pairs than said first target sequence such thatsaid second primer set can amplify said second target sequence in thepresence of some photomodification of said endogenous nucleic acid; e)exposing each of said first and second amplification reaction containingmeans to conditions sufficient to provide amplification products ofdifferent lengths; and f) measuring the quantity of said amplificationproducts in said first and second amplification reaction containingmeans to estimate inactivation of said pathogens, if any, present duringstep (a).
 12. The method of claim 11 wherein said first target sequenceconsists of enough base pairs that photomodification of said endogenousnucleic acid inhibits in part the generation of amplification productsby said first primer set.
 13. A method of estimating inactivation of HIVvirus by measuring the photomodification of endogenous nucleic acid in ablood product having blood cells that contain nucleic acid, comprisingthe steps:a) adding 8-methoxytpsoralen which, upon photoactivation witha spectrum of electromagnetic radiation comprising wavelengths ofapproximately 320 nm and greater, binds covalently with nucleic acid tosaid blood product to photomodify at least some of said endogenousnucleic acid; b) treating said endogenous nucleic acid from said bloodproduct to make it accessible to amplification reagents; c) adding afirst portion of said endogenous nucleic acid of step (b) to a firstamplification reaction containing means in the presence of a firstprimer set which can amplify a first target sequence if said firsttarget sequence is unmodified; d) adding a second portion of saidendogenous nucleic acid of step (b) to a second amplification reactioncontaining means in the presence of a second primer set which canamplify a second target sequence having sufficiently fewer base pairsthan said first target sequence such that said second primer set canamplify said second target sequence in the presence of somephotomodification of said endogenous nucleic acid; e) exposing each ofsaid first and second amplification reaction containing means toconditions sufficient to provide amplification products of differentlengths; and f) measuring the quantity of said amplification products insaid first and second amplification reaction containing means toestimating inactivation of said HIV virus, if any, present during step(a).
 14. The method of claim 13 wherein said first target sequenceconsists of enough base pairs that photomodification of said endogenousnucleic acid inhibits in part the generation of amplification productsby said first primer set.